A novel HEAT repeat protein 5B anaplastic lymphoma kinase fusion in lung adenocarcinoma confers sensitivity to ensartinib.

ALK gene rearrangements occur in approximately 5% of lung adenocarcinomas (ACAs), leading to anaplastic lymphoma kinase (ALK) overexpression and predicting response to targeted therapy. Fluorescence in situ hybridization (FISH) is the standard procedure for detection of ALK rearrangements in lung ACA but requires specialized equipment and expertise. Immunohistochemistry (IHC) for ALK protein overexpression is a promising screening modality, with reports of newer antibodies showing excellent sensitivity and specificity for ALK-rearranged lung ACA. In this study, we analyzed ALK IHC (5A4 clone) in 186 cases from our clinical service and compared it with ALK FISH and EGFR and KRAS mutation status. Twelve cases had concordant ALK protein overexpression and ALK rearrangement by FISH. Three ALK-rearranged cases lacked ALK protein expression. Of these discrepant cases, one had a coexisting EGFR mutation and a subtle atypical ALK rearrangement manifested as a break in the 5' centromeric portion of the FISH probe. One case had a concurrent BRAF mutation. Follow-up testing on a metastasis revealed absence of the ALK rearrangement, with persistent BRAF mutation. In one ALK-rearranged protein negative case, very limited tissue remained for ALK IHC, raising the possibility of false negativity because of protein expression heterogeneity. Importantly, ALK protein expression was detected in one case initially thought not to have an ALK rearrangement. In this case, FISH was falsely negative because of interference by benign reactive nuclei. After correcting for these cases, ALK IHC was 93% sensitive and 100% specific as compared with FISH. ALK IHC improves the detection of ALK rearrangements when used together with FISH, and its use in lung ACA genetic testing algorithms should be considered.

Next-Generation Sequencing Identified a Novel Crizotinib-Sensitive PLB1-ALK Rearrangement in Lung Large-Cell Neuroendocrine Carcinoma

Background:Lung cancer is a leading cause of deaths attributed to cancer worldwide. Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement are commonly found in patients of adenocarcinoma lung against, which targeted therapy is available. In this era of personalized medicine, it is a rationale to detect these molecular alterations in cases of lung carcinomas.Aims:The objectives were to compare the diagnostic efficacy of cytological samples for the detection of EGFR and ALK protein expression using immunocytochemistry in nonsmall cell lung carcinoma.Materials and Methods:We compared 22 cell blocks and biopsies for the detection of EGFR and ALK protein expression by immunohistochemistry (IHC). EGFR IHC was performed using EGFR Receptor (E746-A750 del Specific) (6B6) monoclonal antibody and ALK IHC was done using Ventana anti-ALK (D5F3) monoclonal primary antibody.Results:Two cases were found to be positive; 20 cases were negative for EGFR IHC both in biopsies and cell blocks. ALK IHC was positive in one case; negative in 21 cases. The results of IHC were also concordant for biopsies and cell blocks. The sensitivity and specificity were 100% for immunocytochemical detection of ALK and EGFR in cell blocks with respect to biopsies.Conclusion:We conclude that cell blocks can serve as a potential substitute for biopsies for detection of EGFR and ALK protein by immunocytochemistry, whenever patient presents with effusion and biopsy cannot be done or when tissue is not adequate.

A novel HIP1-ALK fusion variant in lung adenocarcinoma showing resistance to Crizotinib

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) rearrangement are predisposed to molecularly targeted therapies. Proper diagnostic is crucial for quick and correct patients qualification to optimal treatment method. Genetic tests to detect predictive factors could be performed sequentially. After excluding EGFR mutations, abnormal ALK protein expression should be tested using immunohistochemistry (IHC) method. In patients with disrupted ALK expression, the rearrangement of the ALK gene should be confirmed by FISH method. Despite few years of experience in analysis of these predictive factors, there are still problems in interpretation of diagnostic tests results. Especially, some recommendations for ALK IHC diagnosis are not precise. Mutations in EGFR gene were examined using real-time PCR technique in 1,108 formalin-fixed paraffin-embedded (FFPE) tissues, 398 FFPE cell-blocks and 470 cytological specimens of NSCLC. The disrupted ALK protein expression was analysed in 1,100 samples including 782 histological and 306 cytological (cell-blocks) samples using IHC. Twelve materials (1.1%) were non-diagnostic in IHC. ALK gene rearrangement using FISH method was analysed in IHC positive cases. The frequency of EGFR mutations was 8.6%. EGFR mutations occurred significantly more often in females (P=0.00001, χ2=62.732) and in adenocarcinoma cases (P=0.0002, χ2=14.222). The exon 19 deletions (49%) and exon 21 Leu858Arg substitution (38%) were the most common, rare EGFR mutations occurred in 13% of patients. Any expression of abnormal ALK protein was detected in 202 cases (18.57%). ALK gene rearrangement was confirmed in 49 cases (4.5%). ALK gene rearrangement is significantly more common in female than in male (P=0.0105, χ2=6.541). In patients with ALK gene rearrangement, the median percentage of nuclei with ALK rearrangement was only 25.5%. The polysomy (≥4 gene copy number per nuclei) of ALK gene was observed in 39 cases (21.4% of patients with diagnostic result of FISH examination). Median number of ALK gene copy per nuclei was 2.9±0.77. Significant positive correlation between percentage of cells with abnormal ALK expression in IHC test and percentage of nuclei with ALK rearrangement in FISH method was detected (R=0.617, P<0.00001). Significant negative correlation between the number of copies of ALK gene and the percentage of cells with expression of abnormal ALK was observed (R=-0.2004, P<0.05). ALK gene rearrangement was significantly more frequently observed in the material with coarse-grained cytoplasmic and membranous IHC staining than in materials with light cytoplasmic stippling. The occurrence of cytoplasmic stippling correlated with the increase of ALK gene copy number. We indicated that diagnosis of ALK disruption in NSCLC patients should be notably careful using IHC and FISH methods. Recommendations for ALK diagnosis should include the way of interpretation of cases with low percentage of cells with abnormal ALK protein expression in IHC test, character of IHC reaction, and cases with ALK gene polysomy in FISH method.

ALK immunohistochemistry positive, FISH negative NSCLC is infrequent, but associated with impaired survival following treatment with crizotinib

Testing for anaplastic lymphoma kinase (ALK) rearrangement in lung adenocarcinomas: a multicentre study

Anaplastic lymphoma kinase (ALK) rearrangement is a rare feature in advanced non-small cell lung cancer (NSCLC), occurring in around 5% of the patients (1). This oncogenic addiction is more frequent in no/light-smokers, in thyroid transcription factor-1 (TTF1)-positive adenocarcinoma, with cribriform architecture and ring cells (2). ALK rearrangement—most of the time a translocation with a partner gene—induces the formation of a fusion protein with oncogenic properties. The first targeted therapy developed for ALK -rearranged NSCLC was crizotinib, an ALK tyrosine kinase inhibitor (TKI). Crizotinib was proved to be superior to chemotherapy in first-line setting in ALK -rearranged NSCLC, with an overall response rate (ORR) of 74% and a progression-free survival (PFS) of 10.9 months (3). Crizotinib in first-line treatment is now widely used. Other ALK TKIs have been developed, as brigatinib. Results of the phase I-II trial published in 2016 showed in 79 advanced NSCLC patients treated with brigatinib an ORR at 75% [72% in previously crizotinib treated patients (n=71)] and a PFS (median) of 13.2 months (4). Safety profile showed specific lung toxicity, occurring at the beginning of the treatment (median 2 days) in 8% of the patients, with incidence increasing with the dose of brigatinib. The ALTA trial, published in May 2017 in Journal of Clinical Oncology by Kim et al . was a phase II trial that randomized patients with advanced ALK -rearranged NSCLC between 2 doses of brigatinib (90 mg daily and 90 mg daily for 7 days then 180 mg daily) (5). The primary endpoint was ORR, and no comparison between the 2 arms was pre-planned. Assuming an ORR ≥35% with a power of 90% and a bilateral alpha risk at 0.025, the study had to include 109 patients per arm. Patients should have an advanced ALK -rearranged NSCLC with progression with crizotinib and a performance status (PS) between 0 and 2. Brain metastases were allowed only if asymptomatic and with stable doses of steroid treatment. Two-hundred and twenty-two patients were randomized [112 patients arm A (90 mg) and 110 patients arm B (90 mg then 180 mg)]. Both arms were well-balanced, with a median age at 54 years, 57% of women, 31% of Asian, 60% of non-smokers, 97% of adenocarcinoma, 69% of brain metastases. ORR (investigator-assessed) was 45% in arm A and 54% in arm B, and disease-control rate was 82% (arm A) and 86% (arm B). Median time to response was 1.8 months (arm A) and 1.9 months (arm B), and duration of response was 13.8 months (arm A) and 11.8 months (arm B). Median PFS was 9.2 months (arm A) and 12.9 months (arm B). Post-hoc comparison between the 2 arms for PFS showed a clear benefit for arm B, with a hazard ratio (HR) at 0.55 [95% confidence interval (CI), 0.36–0.86]. One-year overall survival (OS) probability was 71% (arm A) and 80% (arm B). As shown in the phase I-II trial, brigatinib had intracranial efficacy. ORR for measurable brain metastases was 42% (arm A, n=26) and 67% (arm B, n=18). For active brain metastases (i.e., metastases without previous brain radiotherapy, or progression after brain radiotherapy), intracranial ORR was 42% (arm A, n=19) and 73% (arm B, n=15). Intracranial PFS (median) was 15.6 months (arm A) and 12.8 months (arm B), with a duration of intracranial response (median) not reached in both arms. In term of safety, no new signal was detected. In both arms, main toxicities (≥10% patients) were nausea (33%/40%), diarrhea (19%/38%), vomiting (24%/23%), headache (28%/27%), decrease appetite (22%/15%) and hypertension (11%/21%). Grade 3–4 toxicities were rare: hypertension (6%/6%), increased creatine phosphokinase (3%/9%) and rash (1%/3%), mainly. Pulmonary early toxicity occurred in 6% of patients (n=14), during the first 2 days of treatment (always at 90mg daily of brigatinib). Grade 3–5 pulmonary toxicities concerned 3% of patients (n=7). Half of the patients (n=7) had successfully continued brigatinib after suspension, whereas 7 patients stopped brigatinib definitely. One patient died from acute respiratory failure (<1%). Multivariate analysis showed that advanced age [odds ratio (OR) =2.10; 95% CI, 1.21–3.65] and time from last crizotinib dose to first brigatinib dose less than 7 days (OR =3.88; 95% CI, 1.10–13.68) were associated with early pulmonary toxicity. Seven percent (arm A) and 20% (arm B) patients had dose reduction, due to increase of creatine phosphokinase, pneumonitis and rash.

The aim of this study was to investigate the diagnostic accuracy of anaplastic lymphoma kinase (ALK) immunohistochemistry (IHC) for ALK gene rearrangement in non-small cell lung cancer (NSCLC) through systematic review, meta-analysis and diagnostic test accuracy review. The current study included 11,806 NSCLC cases in 42 eligible studies. We performed concordance analyses between ALK IHC and fluorescence in situ hybridization (FISH). The diagnostic accuracy of ALK IHC was analyzed based on ALK IHC criteria and antibodies. The overall ALK IHC results were positive in 13.2%. The overall concordance rate between ALK IHC and FISH was 0.950 (95% confidence interval [CI], 0.927-0.966). In the ALK IHC-positive and negative groups, the concordance rates were 0.805 (95% CI 0.733-0.861) and 0.985 (95% CI 0.978-0.990), respectively. The ALK FISH-positive rates were 0.009 (95% CI 0.004-0.023), 0.378 (95% CI 0.217-0.572), 0.628 (95% CI 0.420-0.796) and 0.900 (95% CI 0.840-0.939) in the ALK IHC 0, 1+, 2+ and 3+ groups, respectively. In diagnostic test accuracy review for ALK IHC, the pooled sensitivity and specificity were 0.92 (95% CI 0.89-0.94) and 0.91 (95% CI 0.90-0.91), respectively. The diagnostic odds ratio and the area under the curve on the summary receiver operating characteristic curve were 266.56 (95% CI 110.83-641.14) and 0.983, respectively. Our results suggested that ALK IHC equivocal (score 1+ and 2+) cases should not be considered as IHC-negative in screening for ALK gene rearrangement. Additional detailed criteria for ALK IHC equivocal cases are necessary to determine how to best apply this approach in daily practice.

Next-generation Sequencing Identified a Novel WDPCP-ALK Fusion Sensitive to Crizotinib in Lung Adenocarcinoma

Background. Inflammatory myofibroblastic tumor (IMFT) is a rare mesenchymal neoplasm, mainly driven by anaplastic lymphoma kinase (ALK) rearrangement, which is present in 50% of cases and target for ALK inhibition. For the current project we used tumor material from IMFT patients treated with the ALK/ROS1/MET inhibitor crizotinib in the frame of EORTC 90101, where IMFT patients were attributed to ALK+/- sub-cohorts based on the presence (&amp;gt;15% of cells) or absence (&amp;lt;15%) of ALK rearrangement by fluorescence in situ hybridization (FISH) and/or immunohistochemistry (IHC) [Schöffski at al. Lancet Respir Med. 2018]. The aim of the project was to evaluate the frequency of ALK rearrangement, compare different methods of ALK detection, perform a detailed characterization of ALK rearrangement partners, and to identify other potential molecular drivers with potential pharmacodynamic relevance. Material and Methods. Archival material from 24 IMFT cases, both primary or metastatic samples, was analyzed by IHC using ALK antibodies: ALK1 (DAKO) and ALK-D5F3 (Cell Signalling), and pan-NTRK (Ventana, clone EPR17341). ALK and ROS1 rearrangement were studied by FISH and fusion genes were detected using the Archer CTL Fusion Panel. Results. ALK immunopositivity by ALK1 and ALK-D5F3 was observed in 14 out of 24 cases and 11/21, respectively. ALK rearrangement by FISH was found in 13/23 samples. With Archer, fusion transcripts were identified in 13/20 specimens, all but one involving ALK with 11 different fusion partners. In one case we detected an ETV6-NTRK3 fusion; NTRK positivity was confirmed by IHC. Interestingly, the NTRK fusion case responded to crizotinib, which shows cone anti-NTRK activity [Okamura et al. JCO Precis Oncol 2019]. We did not detect ROS1 rearrangement by FISH in any sample. Two samples with ALK rearrangement by Archer were negative by FISH, with 11% and 0% of cells with ALK split signal, but both showed protein expression with both antibodies used. They responded to crizotinib with either a RECIST 1.1 complete response or stable disease, for 43 and 9 months, respectively. All but one case with ALK rearrangement by Archer were positive by ALK IHC with ALK1 antibody. The only immunonegative sample had an EML4-ALK1 fusion. Two cases were positive with ALK-D5F3 antibody but no ALK positivity was detected using ALK1, and no ALK rearrangement was detected by FISH or Archer panel, suggesting false positive results. Conclusions. ALK rearrangement is the most common driver in IMFT and the fusion can involve multiple partners. ALK1 but not the ALK-D5F3 antibody shows high ALK specificity in IMFT. The Archer CTL Fusion panel is a reliable and sensitive method for detecting fusion transcripts in IMFT, also allowing to detect alternative fusion genes, which may be responsible for the sensitivity to kinase inhibitors. Citation Format: Agnieszka Wozniak, Che-Jui Lee, Tom van Wezel, Jozef Sufliarsky, Hans Gelderblom, Jean-Yves Blay, Maria Debiec-Rychter, Raf Sciot, Judith V.M.G. Bovee, Patrick Schöffski. Detection of molecular drivers in inflammatory myofibroblastic tumor: study on archival tissue from EORTC 90101 “CREATE” phase II clinical trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3191.

P2.09-35 Proposal to Revise VENTANA ALK Scoring Interpretation Guide for Non-Small Cell Lung Carcinoma: Interpretation of ALK Heterogeneity

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC): Results of a multi-centre ALK-testing

Purpose: Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC), identifying patients (pts) who may benefit from ALK tyrosine kinase inhibitors (TKIs). A case is considered positive for rearrangement either based on split 3′ and 5′ signals or loss of the 5′ signal (5′ deletion). We hypothesized that 5′ deletion on FISH may not be completely specific for presence of rearrangement, making the assay vulnerable to false positive results. Methods: An institutional IRB-approved database of NSCLC pts was queried for those with ALK-rearrangements. Clinical characteristics and response to ALK TKIs were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were obtained to further characterize ALK status. Results: Of 1614 NSCLC pts with ALK testing, 97 (6.0%) patients had abnormal ALK FISH: 30 with split signals, 24 with 5′ deletion, 3 with 3′ deletion, 1 with atypical rearrangement, and 39 with details unavailable. Pts with 5′ deletion were older (p = 0.01), had a more extensive smoking history (p = 0.03), and were more likely to harbor mutations in EGFR or KRAS (2 cases vs 0). Pts with 5′ deletion treated with ALK TKIs had a trend toward less tumor shrinkage (p = 0.07). ALK IHC and FISH were discordant in 3 of 21 pts with 5′ deletion and 0 of 26 pts with split signals (p = 0.03). NGS was concordant with ALK IHC, but not FISH, in 2 of 7 samples with 5′ deletions. All three testing modalities were concordant in 3 samples with split signals. Conclusions: NSCLC pts with 5′ deletion on ALK FISH have clinical and molecular features less typical of ALK-rearranged tumors, suggesting an unappreciated risk of false positives with this assay. While the majority of patients with 5′ deletion on ALK FISH harbor a true rearrangement and respond to ALK TKI, confirming these cases with IHC or NGS could reduce the false positive rate. Table 1.Clinicopathologic Features of NSCLC patients with positive ALK FISHSplit Signals (N = 30)5″ Deletion (N = 24)p-valueAge at Diagnosis - yearsp = 0.01MedianRange22-8228-76Age Distribution - No. (%)&amp;lt;40517%14%40-491033%28%50-59827%1042%60-69413%833%&amp;gt;70310%313%Race - No. (%)p = 0.03Caucasian2480%2188%Asian27%28%Other413%14%Smoking History - No. (%)p = 0.03Never Smoker1963%1042%Light Smoker (&amp;lt;10py)620%729%Heavy Smoker517%625%Presence of Co-MutationsEGFR00%14%KRAS00%14%None30100%2292%ALK Immunohistochemistryp = 0.03Positive26100%1886%Negative00%314%Next-Generation SequencingPositive3100%571%Negative00%229% Citation Format: Xin Gao, Lynette M. Sholl, Mizuki Nishino, Jennifer Heng, Pasi A. Janne, Geoffrey R. Oxnard. 5′ deletion on ALK break-apart FISH and risk of false positive results. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4250. doi:10.1158/1538-7445.AM2015-4250

The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported. Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients. Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n=5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9months. ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing.